Cyclic α,α-disubstituted amino acids (dAAs) are powerful tools for stabilizing peptide helices, yet the effects of ring size on helical conformation remain poorly understood. Here, we report the design, synthesis, and structural analysis of template peptides incorporating three cyclic dAAs─1-aminocyclopropane-1-carboxylic acid (Ac3c), 1-aminocyclobutane-1-carboxylic acid (Ac4c), and 1-aminocyclopentane-1-carboxylic acid (Ac5c). Circular dichroism (CD) and infrared (IR) spectroscopy demonstrated that oligomers containing Ac3c-Ac5c residues adopt stable helical structures in solution. X-ray crystallographic studies further revealed the first solid-state structures of Ac3c- and Ac4c-containing peptides, showing well-defined right-handed helices stabilized by intramolecular i → i + 3 and i → i + 4 hydrogen bonds. Comparative analysis highlighted that ring strain in Ac3c weakens its helicogenic effect relative to those of Ac4c and Ac5c, underscoring the importance of ring size in dictating backbone torsion angles and hydrogen-bonding networks. To probe functional implications, template peptides were incorporated into cell-penetrating peptide conjugates (Block3 derivatives). While Ac4c- and Ac5c-containing derivatives retained helicity and supported siRNA internalization, the Ac3c analogue adopted a random conformation and lost activity. These findings establish cyclic dAAs as versatile helix-inducing modules, provide new structural insights into ring-size-dependent helix stabilization, and suggest design principles for peptide-based foldamers and delivery systems.

Monoclonal antibody-based immune checkpoint inhibitors, which have brought breakthrough effects in cancer treatments, are expected to assist in the treatment of viral diseases. However, antibody therapies may cause immune-related side effects, such as inflammation and pneumonia, due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group), namely compound 2 (molecular weight: 456.6), from our library of sulfur-containing protein-protein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1, with the dissociation rate constant (KD) of 77.60 ± 4.44 nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably, oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33, H1N1 subtype) caused a significant increase in the neutralizing antibody titers, as well as recovery from influenza-induced pneumonia. Overall, 2 provides insight for the development of therapeutic drugs against early viral infections, with both virus titer-reducing and antibody titer-boosting effects. Moreover, 2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms.

Cell-penetrating peptides (CPPs) have attracted considerable attention as carriers that facilitate the intracellular delivery of biomacromolecules. In this study, the amphipathic antimicrobial peptide K9L9 was used as a model, and a series of peptides incorporating the non-proteinogenic amino acid α-aminoisobutyric acid (Aib) were designed and synthesized to investigate the relationship between peptide structure, membrane interaction, and cellular uptake behavior. Circular dichroism analysis revealed that K9L9 adopted a β-sheet-like conformation in an aqueous solution, whereas the Aib-containing peptide formed a stable α-helical structure. In liposome leakage assays, peptides containing two Aib residues exhibited enhanced membrane-disruptive activity, whereas the peptide containing four Aib residues exhibited reduced activity. Furthermore, intracellular delivery studies using fluorescein isothiocyanate-dextran (FITC-dextran) demonstrated that delivery efficiency was significantly enhanced when the peptides were preincubated with FITC-dextran, suggesting that peptide-cargo complex formation plays a critical role in the delivery process. In addition, K9L9 exhibited rapid cellular uptake at early time points, whereas the Aib-containing peptides showed a gradual increase in uptake over time, indicating distinct uptake kinetics. These results demonstrate that Aib-induced helix stabilization modulates the membrane interaction and cellular uptake behavior of amphipathic peptides.

We report the development of a redox-responsive system that induces reversible conformational changes in peptides through the design of a seven-membered cyclic α,α-disubstituted α-amino acid with a disulfide bond, 5-amino-1,2-dithiepane-5-carboxylic acid (Dtp). Upon reduction, the disulfide bond in Dtp was cleaved to form thiols, converting Dtp into (2-mercaptoethyl)homocysteine (Mhc), and this process was reversed by oxidation. Dtp-containing peptides predominantly adopted 3 10 -helical conformation in solution, whereas Mhc-containing peptides exhibited a mixture of helical and other conformations. This redox-responsive mechanism allows for precise control over peptide secondary structures, making it a promising approach for designing functional helical peptides capable of acting molecular switches in response to intracellular reductive environments.

Amphipathic helical peptides are common among antimicrobial and cell membrane-permeable peptides, and amino acid substitutions within existing peptides have been an effective means of new peptide drug development. In the present study, we evaluated the effect of introducing a disubstituted amino acid (dAA), which has a stabilizing effect on the helical structure of the amphiphilic peptide C18AA, on the secondary structure of C18AA and its intracellular uptake. Computational analysis was also used to calculate the changes in the thermal stability of the secondary structure caused by substitution to dAAs. The results revealed that peptides that assumed a stable helical structure in aqueous solution showed higher intracellular uptake. It was also revealed that the type and position of the substituted amino acids significantly affected the peptide's secondary structure and intracellular uptake. These results indicate that use of dAAs may be a promising approach to improving the intracellular uptake of existing amphipathic helical peptides.

The decapeptide Boc-(D-Phe-tFPro-Val-Leu-Leu)2—OMe (1) (Boc is tert-butoxycarbonyl, tFPro is 4-trans-fluoro-L-proline D-Phe is D-phenylalanine, Val is valine and Leu is leucine) crystallized in a methanol-solvated form (C68H104F2N10O13·CH4O). Peptide 1 has a sequence similar to gramicidin S (GS) incorporating tFPro. GS is a cyclic peptide, with the D-Phe-Pro unit known as a strong β-turn inducer in previous studies. Thus, it was initially assumed that 1 would bend at the D-Phe6-tFPro7 position, potentially forming a sheet-like structure. However, the structure of 1 was a helix, a surprising finding in GS-related structural studies. A factor enabling this helical formation could be the fluorine–H interactions between tFPro and the aromatic rings of D-Phe residues.

This study explores the formation of a molecular complex between moxifloxacin (MFX) and the artificial sweetener saccharin (SAC) as a strategy to mask the drug's undesirable taste. MFX particles were combined with SAC in a 1:1 ratio using ball milling, a process that facilitated the formation of both cocrystal and coamorphous salts through ionic interactions. Structural analysis of the MFX-SAC complex revealed intermolecular ionic bonds between the amino group of MFX and the sulfonamide group of SAC. Solid-state nuclear magnetic resonance spectroscopy further confirmed that MFX interacts with SAC via ionic bonding. Electronic taste evaluations demonstrated that the MFX-SAC complex not only significantly reduced the bitterness of MFX but also suppressed the overall taste intensity to levels below the quinine reference standard. These findings highlight the potential of molecular complex formation between artificial sweeteners and bitter-tasting drugs as an effective approach for taste modification and masking, offering promising implications for pharmaceutical formulation development.

Using a Phe-incorporated cyclic peptide as a scaffold, we discussed the local control of CH⋯π interactions and the associated flexibility of the Phe side chain by the 4-position substituents and their effects on global conformational equilibrium.

The cyclic peptide cyclo(Val-Leu-Leu-D-Phe-Pro)2 (peptide 1) was specifically designed for structural chemistry investigations, drawing inspiration from Gramicidin S (GS). Previous studies have shown that Pro residues within 1 adopt a down-puckering conformation of the pyrrolidine ring. By incorporating fluoride-Pro with 4-trans/cis-isomers into 1, an up-puckering conformation was successfully induced. In the current investigation, introducing hydroxyprolines with 4-trans/cis-isomer configurations (tHyp/cHyp) into 1 gave cyclo(Val-Leu-Leu-D-Phe-tHyp)2 methanol disolvate monohydrate, C62H94N10O12·2CH4O·H2O (4), and cyclo(Val-Leu-Leu-D-Phe-cHyp)2 monohydrate, C62H94N10O12·H2O (5), respectively. However, the puckering of 4 and 5 remained in the down conformation, regardless of the geometric position of the hydroxyl group. Although the backbone structure of 4 with trans-substitution was asymmetric, the asymmetric backbone of 5 with cis-substitution was unexpected. It is speculated that the anticipated influence of stress from the geometric positioning, which was expected to affect the puckering, may have been mitigated by interactions between the hydroxyl groups of hydroxyproline, the solvent molecules, and peptides.

Conformational freedom-restricted peptides, such as stapled peptides, play a crucial role in the advancement of functional peptide development. We synthesized stapled octapeptides using α-carbocyclic α,α-disubstituted α-amino acids, particularly 3-allyloxy-1-aminocyclopentane-1-carboxylic acid, as the crosslink motifs. The organocatalytic capabilities of the synthesized stapled peptides were assessed in an asymmetric nucleophilic epoxidation reaction because the catalytic activities are known to be proportional to α-helicity. Despite incorporating side-chain crosslinks, the enantioselectivities of the epoxidation reaction catalyzed by stapled octapeptides were found to be comparable to those obtained using unstapled peptides. Interestingly, the stapled peptides using α-carbocyclic α,α-disubstituted α-amino acids demonstrated higher reactivities and stereoselectivities (up to 99% ee) compared to stapled peptides derived from (S)-α-(4-pentenyl)alanine, a commonly used motif for stapled peptides. These differences could be attributed to the increased α-helicity of the former stapled peptide in contrast to the latter, as evidenced by the X-ray crystallographic structures of their N-tert-butoxycarbonyl derivatives.

Cocrystal engineering is a potent strategy for enhancing drug properties, but its application in spray-drying has been limited. Furthermore, concurrently introducing two drugs and realizing simultaneous pharmacological effects at the target site is challenging. This is particularly evident for oral formulations designed for systemic use, but the local administration of drugs at the target site displays the potential to overcome this problem. Herein, we investigated the potential of cocrystallization principles for use in developing dry powder inhaler formulations of anti-tuberculosis drugs. We focused on the formation of cocrystals of 4-aminosalicylic acid (PAS) and isoniazid (INH), which are crucial components of tuberculosis treatment. Via spray-drying, we successfully produced spray-dried particles of PAS-INH that exhibited hydrogen bonding interactions between the molecules. Aerosolization performance evaluation with an Andersen Cascade Impactor (ACI) confirmed the concurrent deposition of both drugs, highlighting the potential of our particle design for use in the co-delivery of PAS-INH. Furthermore, simulated lung dissolution studies using the ACI and Transwell inserts revealed synchronized dissolution patterns, indicating promising prospects for use in effective drug delivery. This innovative approach to tuberculosis therapy has significant implications for improving treatment efficacy and enhancing patient adherence, potentially revolutionizing tuberculosis treatment.

Ascidiacyclamide [cyclo(-Ile-oxazoline-D-Val-thiazole-)2] is a cytotoxic cyclic peptide from ascidian. We examined the potential of the CH⋯π interaction at the diagonal position of ascidiacyclamide by comparing the interactions of Ile, Val, Abu (2-aminobutyric acid) or Ala with Ile, Chg (cyclohexylglycine) or Phg (phenylglycine). In solution, ascidiacyclamides are in a conformational equilibrium between square and folded forms. The CH⋯π interaction is expected to contribute to stabilization of the square form, which enhances the peptides' cytotoxicity. The distances between the alkyl side chain of Xaa and the π-plane of Phg were estimated from the crystal structures. The conformational free energies (ΔG°) determined through NMR-based quantitation indicated remarkable stabilization of the square form upon incorporation of Phg. These observations were consistent with the circular dichroism (CD) spectral measurements. Chemical shift perturbation studies suggested that stabilization of the square form of Phg-incorporated peptides was due to the CH⋯π interaction with the alkyl side chain of Xaa. Greater enthalpic losses were caused during the folding process of Phg-incorporated peptides than Ile- or Chg-incorporated peptides. It is suggested that these enthalpic losses are relevant to the CH⋯π interaction energies, which must be disrupted during folding. In addition, the CH⋯π interactions in the Phg-incorporated peptides increased cytotoxicity.

Although deuterium incorporation into pharmaceutical drugs is an attractive way to expand drug modalities, their physicochemical properties have not been sufficiently examined. This study focuses on examining the changes in physicochemical properties between flurbiprofen (FP) and flurbiprofen-d8 (FP-d8), which was successfully prepared by direct and multiple H/D exchange reactions at the eight aromatic C-H bonds of FP. Although the effect of deuterium incorporation was not observed between the crystal structures of FP and FP-d8, the melting point and heat of fusion of FP-d8 were lower than those of FP. Additionally, the solubility of FP-d8 increased by 2-fold compared to that of FP. Calculation of the interaction energy between FP/FP-d8 and water molecules using the multi-component density functional theory method resulted in increased solubility of FP-d8. These novel and valuable findings regarding the changes in physicochemical properties triggered by deuterium incorporation can contribute to the further development of deuterated drugs.

Ornithine-free Gramicidin S (1, cyclo(Val-Leu-Leu-D-Phe-Pro)2) is a good scaffold for studying β-turn and sheet structures. 4-Fluororide proline was incorporated into 1, and the resulting puckering of Pro was evaluated. Two geometric isomers, trans-4-fluoro-Pro and cis-4-fluoro-Pro, were incorporated into peptides 2 and 3, respectively. 4,4-Difluoro-Pro, which had no isomer, was incorporated into 4. The fluoro-analogues of 2–4 formed sheet and β-turn structures. The "up" forms (Cβ-endo and Cγ-exo) of Pro were found in 2, which is their first observation in Gramicidin S analogues. The "down" forms (Cβ-exo), which have been observed previously in GS analogues, were found in 3 and 4. Energy minimization indicated a 3–4% energy benefit in the "down" form. 4-Fluororide proline was incorporated into Ornithine-free Gramicidin S, and the resulting puckering of Pro (pyrrolidine ring) was evaluated. The "up" forms of Pro were found, which is the first observation in Gramicidin S analogues. Energy minimization indicated a 3–4% energy benefit in the "down" form.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

We have previously reported that cyclo(l-Leu-d-Leu-Aib-l-Leu-d-Leu-Aib) (2), a cyclic hexapeptide consisting of heterochiral l-Leu and d-Leu (l-Leu-d-Leu) residues with achiral 2-aminoisobutyric acid (Aib) residues, forms a figure-8 conformation. In this study, we newly designed cyclo(l-Leu-d-Leu-Aib-d-Leu-l-Leu-Aib)+ (4), an epimer of 2, and examined the conformational differences between 2 and 4 by X-ray crystallographic analysis. Peptide 4 formed a planar cyclic conformation with an antiparallel β-sheet hydrogen-bonding pattern. This investigation demonstrates the potential to manipulate the molecular conformation of cyclic peptides by simply arranging the l- and d-amino acids and emphasizes that diverse conformations can be obtained by using cyclic peptides. Harnessing cyclic peptides as platforms for distinct molecular structures is a promising approach to expanding the chemical space for various applications.

We present an E-selective ring-closing metathesis reaction in α-helical stapled peptides at positions i and i + 4. The use of two chiral carbocyclic α,α-disubstituted α-amino acids, (1S,3S)-Ac5c3OAll and (1R,3S)-Ac5c3OAll, provides a high E-selectivity of a ≤59:1 E:Z ratio, while mixtures with E:Z ratios of 2.1-0.5:1 were produced with standard acyclic (S)-(4-pentenyl)alanine amino acids. A stapled octapeptide composed of (1S,3S)- and (1R,3S)-Ac5c3OAll amino acids showed a right-handed α-helical crystal structure.

Amphipathic cell-penetrating peptides based on the pep-1 sequence were synthesized by replacing the three hydrophilic glutamic acid residues with disubstituted, non-proteinogenic, hydrophobic amino acids. These substitutions facilitated maintenance of the peptides' secondary structure in a helical conformation, even in aqueous solution. Stability against enzymatic degradation was improved through the use of disubstituted amino acids. The resultant peptides exhibited high membrane permeability that remained relatively stable during prolonged incubation times. The results of this study indicate that the use of non-proteinogenic amino acids may be an effective approach to improve the cell membrane permeability for existing amphiphilic peptides.

Cell-penetrating peptides (CPPs) have been attracting attention as tools for intracellular delivery of membrane-impermeant functional molecules. Among the variety of CPPs that have been developed, many are composed of both natural and unnatural amino acids. We previously synthesized α,α-disubstituted α-amino acids (dAAs) containing a five-membered carbocyclic ring in its side chain and revealed the utility of dAAs for the development of novel CPPs. In the present study, we designed a six-membered carbocyclic ring dAA with an amino group on the ring and introduced it into arginine (Arg)-rich peptides to further investigate the value of dAAs for developing CPPs. We also assessed the effects of the size of the dAA carbocyclic ring on cellular uptake of dAA-containing peptides. The stability of the peptide's secondary structure and its membrane permeability were both greater in dAA-containing peptides than in an Arg nonapeptide. However, the number of carbon atoms in the dAA side chain ring had little effect. Nevertheless, these results show the utility of cyclic dAAs in the design of novel CPPs containing unnatural amino acids.

The structure of an ornithine (Orn)-free Gramicidin S (GS) analogue, cyclo(Val-Nle-Leu-D-Phe-Pro)2 (NGS), was studied. Its circular dichroism (CD) spectrum showed that NGS has a structure similar to GS, though the value of [θ] indicated smaller β-turn and sheet populations. This is probably because the Nle side chain could not form intramolecular hydrogen bonds stabilizing the sheet structure. The chemical shift perturbation of αH and JNH-αH were similar in GS and NGS. Three independent NGS molecules formed intramolecular β-sheet structures in crystal. The turn structures of D-Phe-Pro moieties were classed as type II' β-turns, but one part was unclassed. The molecules were arranged in a twisting manner, which resulted in the formation of a helical sheet. Similar structural characteristics were observed previously in a Leu-type, Orn-free GS analogue and in GS trifluoroacetic acid salt.